One-day enzymatic synthesis and purification of UDP-N- [1-14C]acetyl-glucosamine.
UDP-GlcN[1-14C]Ac was synthesized in a single enzymatic reaction from [1-14C]acetate and commercially available precursors on both a microcurie (micromole) and a millicurie (millimole) scale. The reaction was catalyzed by the action of acetyl coenzyme A synthetase, inorganic pyrophosphatase, and the bifunctional Escherichia coli GlmU protein. Within 2 h 86 to 94% reaction is attained, and it approaches 99% completion overnight. GlmU protein was prepared in the form of a fusion suitable for nickel chelate affinity chromatography. Several methods were developed for rapid purification of UDP-GlcN[1-14C]Ac: an HPLC method handled micromole (microcurie) loads. Alternatively, ion exchange chromatography over DOWEX AG1 X-2 using a batch elution procedure was compatible with millimole (millicurie) amounts of radiolabel and yielded both chemically and radiochemically homogeneous UDP-GlcN[1-14C]Ac. These methods allow laboratories to quickly produce and purify microcurie to millicurie quantities of N-acetyl-labeled UDP-GlcNAc by a choice of methods from relatively inexpensive precursors.
Leiting
B
B
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Pryor
K D
KD
Eveland
S S
SS
Anderson
M S
MS
D018528:ATP-Binding Cassette Transporters; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D018528:ATP-Binding Cassette Transporters; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D014537:Uridine Diphosphate N-Acetylglucosamine; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase